Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease

Ongoing inflammation including activation of the complement system is a hallmark of systemic lupus erythematosus (SLE). Antimicrobial neutrophil extracellular traps (NETs) are composed of secreted chromatin that may act as a source of autoantigens typical for SLE. In this study, we investigated how complement interacts with NETs and how NET degradation is affected by complement in SLE patients. We found that sera from a subset of patients with active SLE had a reduced ability to degrade in vitro-generated NETs, which was mostly restored when these patients were in remission. Patients that failed to degrade NETs had a more active disease and they also displayed lower levels of complement proteins C4 and C3 in blood. We discovered that NETs activated complement in vitro and that deposited C1q inhibited NET degradation including a direct inhibition of DNase-I by C1q. Complement deposition on NETs may facilitate autoantibody production, and indeed, Abs against NETs and NET epitopes were more pronounced in patients with impaired ability to degrade NETs. NET-bound autoantibodies inhibited degradation but also further increased C1q deposition, potentially exacerbating the disease. Thus, NETs are a potent complement activator, and this interaction may play an important role in SLE. Targeting complement with inhibitors or by removing complement activators such as NETs could be beneficial for patients with SLE.     

Three dehalogenases and physiological restraints in the biodegradation of haloalkanes by Arthrobacter sp. strain HA1

Arthrobacter sp. strain HA1 utilizes 18 C2-to-C8 1-haloalkanes for growth and synthesizes an inducible 1-bromoalkane debrominase of unknown physiological function (R. Scholtz, T. Leisinger, F. Suter, and A.M. Cook, J. Bacteriol. 169:5016-5021, 1987) in addition to an inducible 1-chlorohexane halidohydrolase which dehalogenates some 50 substrates, including alpha, omega-dihaloalkanes. alpha, omega-Dihaloalkanes were utilized by cultures of strain HA1 under certain conditions only. C9 and C8 homologs prevented growth. At suitable concentrations, C7-to-C5 homologs could serve as sole sources of carbon and energy for growth. C4 and C3 homologs could be utilized only in the presence of a second substrate (e.g., butanol), and the C2 homolog was not degraded. Kinetics of growth and substrate utilization indicated that cells of strain HA1 growing in butanol-salts medium could be used to test whether compounds induced the 1-chlorohexane halidohydrolase. No gratuitous induction of synthesis of the enzyme was observed. Many enzyme substrates (e.g., bromobenzene) did not induce synthesis of the enzyme, though the enzyme sequence to degrade the product (phenol) was present. Some inducers (e.g., bromomethane) were enzyme substrates but not growth substrates. In an attempt to find a physiological role for the 1-bromoalkane debrominase, we observed that several long-chain haloaliphatic compounds (greater than C9; e.g., 1-bromohexadecane and 1-chlorohexadecane) were utilized for growth and that induced cells could dehalogenate several 1-haloalkanes (at least C4 to C16). The dehalogenation of the long-chain compounds could not be assayed in the cell extract, so we presume that a third haloalkane dehalogenase was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three dehalogenases and physiological restraints in the biodegradation of haloalkanes by Arthrobacter sp. strain HA1.

Arthrobacter sp. strain HA1 utilizes 18 C2-to-C8 1-haloalkanes for growth and synthesizes an inducible 1-bromoalkane debrominase of unknown physiological function (R. Scholtz, T. Leisinger, F. Suter, and A.M. Cook, J. Bacteriol. 169:5016-5021, 1987) in addition to an inducible 1-chlorohexane halidohydrolase which dehalogenates some 50 substrates, including alpha, omega-dihaloalkanes. alpha, omega-Dihaloalkanes were utilized by cultures of strain HA1 under certain conditions only. C9 and C8 homologs prevented growth. At suitable concentrations, C7-to-C5 homologs could serve as sole sources of carbon and energy for growth. C4 and C3 homologs could be utilized only in the presence of a second substrate (e.g., butanol), and the C2 homolog was not degraded. Kinetics of growth and substrate utilization indicated that cells of strain HA1 growing in butanol-salts medium could be used to test whether compounds induced the 1-chlorohexane halidohydrolase. No gratuitous induction of synthesis of the enzyme was observed. Many enzyme substrates (e.g., bromobenzene) did not induce synthesis of the enzyme, though the enzyme sequence to degrade the product (phenol) was present. Some inducers (e.g., bromomethane) were enzyme substrates but not growth substrates. In an attempt to find a physiological role for the 1-bromoalkane debrominase, we observed that several long-chain haloaliphatic compounds (greater than C9; e.g., 1-bromohexadecane and 1-chlorohexadecane) were utilized for growth and that induced cells could dehalogenate several 1-haloalkanes (at least C4 to C16). The dehalogenation of the long-chain compounds could not be assayed in the cell extract, so we presume that a third haloalkane dehalogenase was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Novel Peptide Can Mimic Extracellular Fibrinogen-Binding Protein to Block the Activation of Complement System

Extracellular fibrinogen-binding protein (Efb) of Staphylococcus aureus (S. aureus) is a bi-functional protein, which can specifically bind fibrinogen with its N terminus and inhibit deposition of C3b on the surface of S. aureus with its C terminus. Here, we screened the epitopes of Efb using phage display. Four peptides with consensus motif were screened. This consensus motif was identical to C terminus (161–164) of Efb. In the further investigation, it was found the synthesized peptide EC1 (154–165aa of Efb) could specifically bind C3/C3b and subsequently to block the activation of complement. Meanwhile, EC1 could inhibit the interaction between Efb and C3/C3b. Moreover, the interaction between the mutant protein of EmC1 (Efb without EC1) and C3 was decreased. And, the effect on the complement system of the mutant protein was dramatically declined compared with Efb. Our finding suggested that the peptide EC1 could mimic Efb to block complement system activation via binding C3.     

Immunoenzymoassay of the hyaluronic acid-hyaluronectin interaction: application to the detection of hyaluronic acid in serum of normal subjects and cancer patients

The binding of a hyaluronic acid-binding glycoprotein, hyaluronectin (HN), isolated from human brain, to hyaluronic acid (HA) was investigated with the enzyme-linked immunosorbent assay technique using plastic microtest plates coated with a 50 mg/liter solution of HA in 0.1 M bicarbonate. Optimum conditions for HN binding to HA were in 0.2 M NaCl buffered with 0.1 M sodium phosphate at pH 7. An assay for HA in solution was set up exploiting the fact that HN binding could be inhibited by soluble HA. HA was preincubated for 1 h in a test tube with a 30-ng/ml HN solution (v/v) in the buffer containing 0.1% bovine serum albumin. Incubation on HA-coated microtest plate lasted 4 h and maximum sensitivity was achieved when incubation was carried out at 4 degrees C. HN bound to the plate was revealed by means of alkaline phosphatase-conjugated anti-HN antibodies. The test was used to measure HA inhibitory activity after depolymerization by ferrous ions. No difference was found between inhibitory activity or smaller fragments and that of high-molecular-weight HA. The assay was applied to determination of HA in sera. Specificity was demonstrated by Streptomyces hyaluronidase digestion of reactive material in sera. Other glycosaminoglycans did not interfere with the assay. Recovery of HA was good and intra- and interassay variation coefficients were 6 +/- 2.2 and 12%. In 103 blood donor sera, HA was found at 22.4 +/- 16.7 micrograms/liter. HA was elevated in most of the cancer patient sera tested.     

Complement C2 receptor inhibitor trispanning and the beta-chain of C4 share a binding site for complement C2

Complement C2 receptor inhibitor trispanning (CRIT) of the Schistosoma parasite binds human C2 via the C2a segment. The receptor in vivo functions as C2 decoy receptor by directly competing with C4b for binding to C2. As a result, CRIT is able to limit the extent of classical pathway (CP) C3 convertase formation. We report that the CRIT-extracellular domain 1 (ed1) peptide inhibits CP-mediated complement activation with an ICH(50) of approximately 0.1 microM, the C-terminal 11 aa of CRIT-ed1, named H17, even more effectively. The beta-chain region F222-Y232 of C4 shares 55% identity and 73% similarity with H17. Peptides based on this region also inhibit CP in a dose-dependent manner. As further evidence of C2 binding we showed CRIT-ed1 peptides and homologous C4 beta-chain peptides to inhibit complement in C2 hemolytic assays. We have predicted C4 beta-c F222-Y232 as a C2 binding site which we have termed the CRIT-ed1 domain, and the sequence [F/H]EVKX(4/5)P as a consensus C2-binding sequence. Anti-CRIT-ed1 cross-reacts with the C4 beta-chain and F222EVKITPGKPY232 appears to be the key epitope recognized by this Ab. Furthermore, anti-CRIT-ed1 was found to inhibit CP activation in a total hemolytic assay. We believe that Schistosoma CRIT-ed1, as well as C4 beta-chain peptides based on the CRIT-ed1 domain, function as interface peptides. These peptides, based on C2-binding sequences in CRIT, or C4, competitively inhibit the binding of C2 to C4b and thus limit the activation of C. The C4 peptides, unlike CRIT-ed1, did not inhibit the cleavage of C2 by C1s.     

Three pentraxins C-reactive protein,serum amyloid p component and pentraxin 3 mediate complement activation using Collectin CL-P1

BackgroundPentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition.MethodsWe used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay.ResultsWe demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation.ConclusionOur current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation.General significanceCL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.     

The importance of sialic acid,pH and ion concentration on the interaction of uromodulin and complement factor H

Uromodulin (UMOD) can bind complement factor H (cFH) and inhibit the activation of complement alternative pathway (AP) by enhancing the cofactor activity of cFH on degeneration of C3b. UMOD, an N-glycans-rich glycoprotein, is expressed in thick ascending limb of Henle's loop where the epithelia need to adapt to gradient change of pH and ion concentration. ELISA-based cofactor activity of cFH and erythrocytes haemolytic assay was used to measure the impact of native and de-glycosylated UMOD on the functions of cFH. The binding assay was performed under different pH and ion concentrations, using ELISA. The levels of sialic acid on UMOD, from healthy controls and patients with chronic kidney disease (CKD), were also detected by lectin-ELISA. It was shown that removal of glycans decreased the binding between UMOD and cFH and abolished the ability of enhancing C3b degradation. In acidic condition, the binding became stronger, but it reduced as sodium concentration increased. A significant decrease of α-2,3 sialic acids on UMOD was observed in CKD patients compared with that of healthy individuals. The sialic acids on UMOD, local pH and sodium concentration could impact the binding capacity between UMOD and cFH and thus regulate the activation of complement AP.     

Complement activation by measles virus cytotoxic antibodies: alternative pathway C activation by hemagglutination-inhibition antibodies but classical activation by hemolysin antibodies.

Antibodies against measles virus hemagglutinating (HA particles and hemolysin were shown to activate C differently. HA antibodies of rabbit or human origin activated C via the alternative pathway in cytotoxicity against chronically measles-infected cells. This cytotoxicity was expressed in C-4 deficient guinea-pig C or in rabbit C in the presence of 3 mM EGTA (ethylene-glycol-tetraacetic-acid) but not in 3 mM EDTA (ethylene-diamine-tetraacetic-acid). In contrast, human hemolysin antibodies activated C only via the classical way. F (ab')2 fragments from rabbit or human anti-HA IgG antibodies were as efficient in C activation via the alternative pathways as intact IgG antibodies with a corresponding hemagglutination-inhibition titer.     

Hyaluronic Acid Oligosaccharides Suppress TLR3-Dependent Cytokine Expression in a TLR4-Dependent Manner

The release of endogenous molecules from the skin after injury has been proposed to influence inflammation. Recent studies have found that pro-inflammatory signals can be generated by damaged endogenous self-RNA, and this event is detected by TLR3. Conversely, release of endogenous fragments of hyaluronic acid (HA) after injury has been proposed to inhibit LPS induced inflammation driven by TLR4. In this study we investigated if HA oligomers could also influence inflammation mediated by TLR3. A tetramer form of HA (oligo-HA) was added to MH-S cells (mouse alveolar macrophage cell line) that were then activated by poly(I:C). ELISA analysis of culture supernatants showed that the presence of oligo-HA suppressed the poly(I:C) induced release of IL-6 and TNFα. IL-6 mRNA expression was also suppressed as measured by quantitative RT-PCR. To determine the mechanism of action for oligo-HA to inhibit poly(I:C), macrophages derived from wild-type (WT), Tlr2−/− or Tlr4−/− mice were treated with oligo-HA and poly(I:C). Similar to WT cells, Tlr2−/− macrophages were inhibited by oligo-HA and retained suppression of cytokine release. In contrast, Tlr4−/− macrophages lost the capacity to be suppressed by oligo-HA. An increase in Traf1 (TLR negative regulator) mRNA was observed after oligo-HA treatment of WT but not in Tlr4−/− macrophages, and oligo-HA did not suppress cytokine responsiveness in Traf1−/− macrophages. These results show that oligo-HA acts through TLR4 and TRAF1 to inhibit TLR3-dependent inflammation. This observation illustrates the complex immunomodulatory action of endogenous products released after injury.     

Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity.

The influenza virus hemagglutinin (HA) contains a cytoplasmic domain that consists of 10 to 11 amino acids, of which five residues have sequence identity for 10 of 13 HA subtypes. To investigate properties of these conserved residues, oligonucleotide-directed mutagenesis was performed, using an HA cDNA of influenza virus A/Udorn/72 (H3N2) to substitute the conserved cysteine residues with other residues, to delete the three C-terminal conserved residues, or to remove the entire cytoplasmic domain. The altered HAs were expressed in eukaryotic cells, and the rates of intracellular transport were examined. It was found that substitution of either conserved cysteine residue within the cytoplasmic domain did not affect the rate of intracellular transport, whereas deletion of residues within the C-terminal domain resulted in delayed cell surface expression. All the altered HAs were biologically active in hemadsorption and fusion assays. To investigate whether the wild-type HA and HAs with altered cytoplasmic tails could complement the influenza virus temperature-sensitive transport-defective HA mutant A/WSN/33 ts61S, the HA cDNAs were expressed by using a transient expression system and released virus was assayed by plaque analysis. The wild-type HA expression resulted in a release of approximately 10(3) PFU of virus per ml. Antibody neutralization of complemented virus indicated that the infectivity was due to incorporation of wild-type H3 HA into ts61S virions. Sucrose density gradient analysis of released virions showed that each of the HA cytoplasmic domain mutants was incorporated into virus particles. Virions containing HAs with substitution of the cysteine residues in the cytoplasmic domain were found to be infectious. However, no infectivity could be detected from virions containing HAs that had deletions in their cytoplasmic domains. Possible roles of the HA cytoplasmic domain in forming protein-protein interactions in virions and their involvement in the initiation of the infection process in cells are discussed.     

猪链球菌2型一种新的免疫原性蛋白的鉴定及特性分析

利用免疫蛋白组学方法,鉴定出链球菌2型(Streptococcus suis type2,SS2)江苏分离株HA9801具有免疫反应性的蛋白HM3。应用同源性比对、信号肽预测、跨膜区预测及亚定位预测等生物信息学方法对该蛋白进行分析,结果显示:同源性最高的蛋白为屎肠球菌胞外溶解物结合蛋白(41%);蛋白序列中含有信号肽结构;7-24位氨基酸为该蛋白的跨膜区;预测蛋白定位为除胞质外的未定位置。PCR扩增出该蛋白的一段基因定向克隆到表达载体pET-32a( )中并转化入BL21(DE3)宿主菌。重组菌经IPTG诱导后的SDS-PAGE图谱在46kDa处出现融合蛋白的条带。Westernblot表明,此融合蛋白可被SPF微型猪抗SS2血清所识别,提示该蛋白可作为该菌的亚单位疫苗的候选物。     

The purification and properties of the second component of guinea-pig complement.

A method has been developed for the purification to homogeneity of guinea-pig complement component C2. Contrary to previous reports, guinea-pig C2 is a single polypeptide chain with apparent mol.wt. of 102000, the same as human C2. It is cleaved by C1s to yield fragments C2a (apparent molwt. 74000) and C2b (apparent mol.wt. 34000). The amino acid composition and N-terminal sequences of these fragments are similar to those of human C2a and C2b. Human and guinea-pig C2 show more extensive sequence homology to Factor B than previously identified. The known homology around the sites of cleavage by C1s and Factor D has now been extended by a stretch of ten identical or conservatively substituted residues. Sequence homology has now been identified at the N-terminal of C2b and Factor Ba. The properties of the classical-pathway C3 convertases assembled from human C4b, C1s and human or guinea-pig C2 have been compared. The rates of cleavage of human and guinea-pig C2 by C1s (and therefore the rates of assembly of the C3 convertases) are similar. The rate of decay of the activity of the C3 convertase formed from guinea-pig C2 is 10-fold lower than for human C2. This greater stability reflects a higher affinity of guinea-pig C2a for human C4b. The presence of C2b is not necessary for C3 convertase activity.     

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.     

Mortalin/GRP75 Binds to Complement C9 and Plays a Role in Resistance to Complement-dependent Cytotoxicity

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.     

Formation of influenza virus particles lacking hemagglutinin on the viral envelope.

We investigated the intracellular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (ts) mutants (ts134 and ts61S) of influenza virus A/WSN/33. We found that at the nonpermissive temperature (39.5 degrees C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK cells were infected with these mutant viruses, they produced noninfectious virus particles at 39.5 degrees C. The efficiency of particle formation at 39.5 degrees C was essentially the same for both wild-type (wt) and ts virus-infected cells. When compared with the wt virus produced at either 33 or 39.5 degrees C or the ts virus formed at 33 degrees C, these noninfectious virus particles were lighter in density and lacked spikes on the envelope. However, they contained the full complement of genomic RNA as well as all of the structural polypeptides of influenza virus with the exception of HA. In these spikeless particles, HA could not be detected at the limit of 0.2% of the HA present in wt virions. In contrast, neuraminidase appeared to be present in a twofold excess over the amount present in ts virus formed at 33 degrees C. These observations suggest that the presence of HA is not an obligatory requirement for the assembly and budding of influenza virus particles from infected cells. The implications of these results and the possible role of other viral proteins in influenza virus morphogenesis are discussed.     

Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells.

Rat liver endothelial cells in primary cultures at 7 degrees C bind radioactively labelled sodium hyaluronate (HA; Mr 400 000) specifically and with high affinity (Kd = 6 X 10(-11) M). Maximal binding capacity is approx. 10(4) molecules per cell. Inhibition experiments with unlabelled HA and oligosaccharides from HA indicate that each molecule is bound by several receptors acting co-operatively and that the single receptor recognizes a tetra- or hexa-saccharide sequence of the polysaccharide. At 37 degrees C the liver endothelial cells endocytose the HA. The process combines the features of a receptor-mediated and a fluid-phase endocytosis. The rate of internalization does not show any saturation with increasing HA concentration, but is approximately proportional to the polysaccharide concentration at and above the physiological concentration. At 50 micrograms of free HA/l each liver endothelial cell accumulates 0.1 fg of the polysaccharide/min. Fluorescent HA accumulates in perinuclear granules, presumably lysosomes. Degradation products from HA appear in the medium about 30 min after addition of the polysaccharide to the cultures. The radioactivity from HA containing N-[3H]acetyl groups or 14C in the sugar rings is recovered mainly as [3H]acetate and [14C]acetate respectively. Estimations of the capacity of liver endothelial cells to internalize and degrade HA in vitro indicate that these cells may be primarily responsible for the clearance of HA from human blood in vivo.     

Genotoxicity of naturally occurring hydroxyanthraquinones

A variety of structurally related hydroxyanthraquinones (HA) were investigated in a test battery for the evaluation of mutagenicity and cell-transforming activity. The tests were: (1) the Salmonella typhimurium mutagenicity assay, (2) the V79-HGPRT mutagenicity assay, (3) the DNA-repair induction assay in primary rat hepatocytes and (4) the in vitro transformation of C3H/M2 mouse fibroblasts. In Salmonella, most of the tested compounds were mutagenic in strain TA1537, but only a few were active in other strains. Among these were HA with a hydroxymethyl group, such as lucidin and aloe-emodin. In V79 cells, only HA with 2 hydroxy groups in the 1,3 positions (1,3-DHA, purpurin, emodin) or with a hydroxymethyl sidechain (lucidin and aloe-emodin) were mutagenic. The compounds found to be active in V79 cells were also active in the DNA-repair assay and in the C3H/M2 transformation assay. Thus, it appears that the genotoxicity of HA is dependent on certain structural requirements.     

Immune evasion of Moraxella catarrhalis involves ubiquitous surface protein A-dependent C3d binding

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1(299-452) and UspA2(165-318). The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.